The present study was initiated to understand the mechanism of thyroxine binding by prealbumin (PA) and thyroxine binding globulin (TBG). When bound to PA, the spin label analogs of thyroxine and dansylsulfonamide became highly immobilized, indicating a constrained structure of its binding domain. The 2T" values of thyroxine spin label bound to PA and TBG were found to be 64 G and 45 G respectively. This observation suggests that the thyroxine binding site in TBG is less rigid than that in PA.